We report the ability of cellulose to support cells without the use of matrix ligands on the surface of the material, thus creating a two-component system for tissue engineering of cells and materials. Sheets of bacterial cellulose, grown from a culture medium containing Acetobacter organism were chemically modified with glycidyltrimethylammonium chloride or by oxidation with sodium hypochlorite in the presence of sodium bromide and 2,2,6,6-tetramethylpipiridine 1-oxyl radical to introduce a positive, or negative, charge, respectively. This modification process did not degrade the mechanical properties of the bulk material, but grafting of a positively charged moiety to the cellulose surface (cationic cellulose) increased cell attachment by 70% compared to unmodified cellulose, while negatively charged, oxidised cellulose films (anionic cellulose), showed low levels of cell attachment comparable to those seen for unmodified cellulose. Only a minimal level of cationic surface derivitisation (ca 3% degree of substitution) was required for increased cell attachment and no mediating proteins were required. Cell adhesion studies exhibited the same trends as the attachment studies, while the mean cell area and aspect ratio was highest on the cationic surfaces. Overall, we demonstrated the utility of positively charged bacterial cellulose in tissue engineering in the absence of proteins for cell attachment.

Tissue engineering is a rapidly advancing field in regenerative medicine, with much research directed towards the production of new biomaterial scaffolds with tailored properties to generate functional tissue for specific applications. Recently, principles of sustainability, eco-efficiency and green chemistry have begun to guide the development of a new generation of materials, such as cellulose, as an alternative to conventional polymers based on conversion of fossil carbon (e.g., oil) and finding technologies to reduce the use of animal and human derived biomolecules (e.g., foetal bovine serum). Much of this focus on cellulose is due to it possessing the necessary properties for tissue engineering scaffolds, including biocompatibility, and the relative ease with which its characteristics can be tuned through chemical modification to adjust mechanical properties and to introduce various surface modifications. In addition, the sustainability of producing and manufacturing materials from cellulose, as well as its modest cost, makes cellulose an economically viable feedstock. This review focusses specifically on the use of modified cellulose materials for tissue culturing applications. We will investigate recent techniques used to promote scaffold function through physical, biochemical and chemical scaffold modifications, and describe how these have been utilised to reduce reliance on the addition of matrix ligands such as foetal bovine serum.

Combining surface chemical modification of cellulose to introduce positively charged trimethylammonium groups by reaction with glycidyltrimethylammonium chloride (GTMAC) allowed for direct attachment of mammalian MG-63 cells, without addition of protein modifiers, or ligands. Very small increases in the surface charge resulted in significant increases in cell attachment: at a degree of substitution (DS) of only 1.4%, MG-63 cell attachment was > 90% compared to tissue culture plastic, whereas minimal attachment occurred on unmodified cellulose. Cell attachment plateaued above DS of ca. 1.85% reflecting a similar trend in surface charge, as determined from ζ-potential measurements and capacitance coupling (electric force microscopy). Cellulose film stiffness was modulated by cross linking with glyoxal (0.3–2.6% degree of crosslinking) to produce a range of materials with surface shear moduli from 76 to 448 kPa (measured using atomic force microscopy). Cell morphology on these materials could be regulated by tuning the stiffness of the scaffolds. Thus, we report tailored functionalised biomaterials based on cationic cellulose that can be tuned through surface reaction and glyoxal crosslinkin+g, to influence the attachment and morphology of cells. These scaffolds are the first steps towards materials designed to support cells and to regulate cell morphology on implanted biomaterials using only scaffold and cells, i.e. without added adhesion promoters.

3D foam scaffolds were produced in a “bottom-up” approach from lyophilised cationic cellulose nanofibril (CCNF) dispersions and emulsions (CCNF degree of substitution 23.0 ± 0.9%), using a directional freezing/lyophilisation approach, producing internal architectures ranging from aligned smooth walled micro channels, mimicking vascularised tissue, to pumice-like wall textures, reminiscent of porous bone. The open, highly porous architecture of these biomimetic scaffolds included mesopores within the walls of the channels. A combination of SEM and NMR cryoporometry and relaxometry was used to determine the porosity at different length scales: CCNF foams with aligned channels had an average macropore (channel) size of 35 ± 9 μm and a mesopore (wall) diameter of 26 ± 2 nm, while CCNF foams produced from directional freezing and lyophilisation of Pickering emulsions had mesoporous walls (5 ± 3 μm) in addition to channels (54 ± 20 μm). Glyoxal crosslinking both enhanced robustness and stiffness, giving Young's moduli of 0.45 to 50.75 MPa for CCNF foams with degrees of crosslinking from 0 to 3.04 mol%. Porosity and channels are critical scaffold design elements for transport of nutrients and waste products, as well as O₂/CO₂ exchange. The viability of MG-63 cells was enhanced on crosslinked, mechanically stiff scaffolds, indicating that these exquisitely structured, yet robust, foams could provide biomaterial scaffolds suitable for industrial applications requiring 3D cell culturing.

The structure of cationic cellulose nanofibril gels and changes in the nanoparticle form with varying chemical modification are revealed by neutron scattering, rheological and ¹H NMR T₁ relaxation time analyses.